Abstract
Aldo-keto reductase (AKR) 1C2 is a human, cytosolic enzyme that has an important role in the deactivation of the potent androgen dihydrotestosterone (DHT). AKR1C2 can regulate the extent and duration of activation of the androgen receptor by catalyzing the reduction of DHT to the less potent receptor ligand 3α-diol. In this study, we functionally characterize in vitro the effect of 11 naturally occurring nonsynonymous single nucleotide polymorphisms on the ability of AKR1C2 to reduce DHT to 3α-diol. The wild-type and variant enzymes were expressed using a transfected insect cell system, and their kinetic activities were measured using both a specific fluorogenic probe and DHT as substrates. This functional characterization demonstrates that several variant AKR1C2 proteins have significantly reduced or altered reductase activities as shown by their measured kinetic parameters. Data from our two separate in vitro studies revealed significant reductions in Vmax for two variants (F46Y and L172Q) and significantly lower apparent Km values for three variants (L172Q, K185E, and R258C) compared with the wild type. These results provide evidence that several naturally occurring nonsynonymous single nucleotide polymorphisms in AKR1C2 result in reduced enzyme activities. These variant AKR1C2 alleles may represent one factor involved in the variable degradation of DHT in vivo.
Footnotes
-
This work was supported by funding from the Canadian Institutes of Health Research. R.H.T. was supported by a studentship from Merck Research Labs, Inc.
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.109.150995.
-
ABBREVIATIONS: AKR, aldo-keto reductase; DHT, 5α-dihydrotestosterone; 3α-diol, 5α-androstane-3α,17β-diol; 3β-diol, 5α-androstane-3β,17β-diol; UGT, UDP glucuronosyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ESF AF, Expression System Formula Animal Free; MES, 2-(N-morpholino)ethanesulfonic acid; CLint, apparent intrinsic clearance; SNP, single-nucleotide polymorphism; HSD, hydroxysteroid dehydrogenase; WT, wild type; FL, fluorescence units.
-
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received January 14, 2009.
- Accepted March 2, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|